Anti-ceramide antibodies

ABSTRACT

Monoclonal antibodies directed to ceramide that inhibit apoptosis are disclosed. Humanized and scFv versions of the antibodies are also disclosed. Methods for prevention or treatment of apoptosis in a subject by administration of the anti-ceramide antibodies are disclosed.

CROSS-REFERENCE TO PRIOR APPLICATIONS

This application claims priority of U.S. Provisional Application No.62/034,453, filed on Aug. 7, 2014.

FIELD

This application generally relates to antibodies that inhibit celldeath. In particular, the invention relates to inhibition of cell deathwith antibodies directed to ceramide.

BACKGROUND

Acute Radiation Syndrome (ARS) (sometimes known as radiation toxicity orradiation sickness) is an acute illness caused by irradiation of a largeportion of the body by a high dose of penetrating radiation; such ashigh energy X-rays, gamma rays, and neutrons; in a very short period oftime, for example, within a matter of minutes. The major cause of thissyndrome is depletion of immature parenchymal stem cells in specifictissues. Examples of people who suffered from ARS are the survivors ofthe Hiroshima and Nagasaki atomic bombs, the firefighters that firstresponded after the Chernobyl Nuclear Power Plant event in 1986, andsome unintentional exposures to sterilization irradiators. In general,the radiation dose for the induction of ARS is large (i.e., greater than0.7 Gray (Gy) or 70 rads), although mild symptoms may be observed withdoses as low as 0.3 Gy or 30 rads.

Radiation gastrointestinal (GI) syndrome will usually occur with a dosegreater than approximately 10 Gy (1000 rads) although some symptoms mayoccur as low as 6 Gy or 600 rads. Survival is extremely unlikely withthis syndrome due to the destructive and irreparable changes in the GItract and bone marrow. Radiation GI Syndrome typically can be dividedinto three stages. The prodromal stage manifests within several hoursafter exposure and symptoms include anorexia, severe nausea, vomiting,cramps, and diarrhea. The latent stage begins after about two days andthe patient may appear and feel well, however, cells lining the GItract, as well as stem cells in the bone marrow, are dying. Less thanone week after exposure, the manifest illness stage begins, withsymptoms including malaise, anorexia, severe diarrhea, fever,dehydration, and electrolyte imbalance. Death usually occurs within 2weeks as a result of infection, dehydration, and electrolyte imbalance.

In addition to the treatment of acute radiation syndrome, bone marrowtransplantation is currently used to treat a number of malignant andnon-malignant diseases including acute and chronic leukemias, myelomas,solid tumors. However, bone marrow transplantation frequently evokes avariety of immune responses in the host, which results in rejection ofthe graft or graft-versus-host disease (hereinafter, referred to as“GvHD”). The conditioning regimen required prior to transplantation,designed to ablate or suppress the patient's immune system, renders thepatient susceptible to neoplastic relapse or infection. Recent use ofunrelated and HLA non-identical donors has unfortunately increased theincidence of GvHD. While removal of T cells from the donor marrow graftameliorates GvHD, this strategy increases graft failure rates andmarkedly diminishes the therapeutically-beneficial graft-versus-tumoreffect. As such, overall survival does not improve. Further, despitestrong pre-clinical data, attempts to improve GvHD outcomes bydiminishing inflammatory cytokine action by adding TNF antagonists tocorticosteroids, the standard of care for acute GvHD, has providedlimited therapeutic benefit.

Thus, there is an urgent need for alternative strategies to reduce theincidence and severity of Radiation GI Syndrome and GvHD.

SUMMARY

One aspect of the present application is directed to an anti-ceramideantibody, or an antigen-binding fragment thereof, comprising: a heavychain variable region CDR1 of 10 amino acids comprising a Gly in the 1stposition from the N-terminal, a Tyr or Phe in the 2nd position from theN-terminal, a Phe or Leu in the 4th position from the N-terminal, and aThr or His in the 6th position from the N-terminal and a His or Asn inthe 10th position from the N-terminal; a heavy chain variable regionCDR2 of 16-17 amino acids comprising a Asn or Ile in the 2nd positionfrom the N-terminal, a Phe or Ser in the 4th position from theN-terminal, a Thr in the 9th position from the C-terminal, a Tyr in the7th position from the C-terminal, an Asn in the 6th position from theC-terminal, a Lys or Ala in the 2nd and 4th positions from theC-terminal; a heavy chain variable region CDR3 of 7 to 11 amino acidscomprising a Tyr or Thr at the 4th position from the N-terminal; a lightchain variable region CDR1 of 10-16 amino acids comprising an Ala or Serin the 2nd position from the N-terminal, a Ser in the 3rd position fromthe N-terminal, a Ser or Asp in the 5th position from the N-terminal,and a Tyr, Ser or Phe in the 3th position from the C-terminal; a lightchain variable region CDR2 of 7 amino acids comprising a Ser or Asn inthe 3rd position from the N-terminal, a Lys or Ser in the 5th positionfrom the N-terminal and a Ser or Asp in the 7th position from theN-terminal; and a light chain variable region CDR3 of 9 amino acidscomprising a Gln, Leu or Trp in the 1st position from the N-terminal, aGln in the 2nd position from the N-terminal, a Pro in the 7th positionfrom the N-terminal and a Thr in the 9th position from the N-terminal.

Another aspect of the present application is directed to ananti-ceramide single-chain variable fragment (scFv) that binds to thesame antigenic determinant as the anti-ceramide antibody of the presentapplication. The scFv comprises: a heavy chain variable region CDR1 of10 amino acids comprising a Gly in the 1st position from the N-terminal,a Tyr or Phe in the 2nd position from the N-terminal, a Phe or Leu inthe 4th position from the N-terminal, and a Thr or His in the 6thposition from the N-terminal and a His or Asn in the 10th position fromthe N-terminal; a heavy chain variable region CDR2 of 16-17 amino acidscomprising an Asn or Ile in the 2nd position from the N-terminal, a Pheor Ser in the 4th position from the N-terminal, a Thr in the 9thposition from the C-terminal, a Tyr in the 7th position from theC-terminal, an Asn in the 6th position from the C-terminal, a Lys or Alain the 2nd and 4th positions from the C-terminal; a heavy chain variableregion CDR3 of 7 to 11 amino acids comprising a Tyr or Thr at the 4thposition from the N-terminal; a light chain variable region CDR1 of10-16 amino acids comprising an Ala or Ser in the 2nd position from theN-terminal, a Ser in the 3rd position from the N-terminal, a Ser or Aspin the 5th position from the N-terminal, and a Tyr, Ser or Phe in the3th position from the C-terminal; a light chain variable region CDR2 of7 amino acids comprising a Ser or Asn in the 3rd position from theN-terminal, a Lys or Ser in the 5th position from the N-terminal and aSer or Asp in the 7th position from the N-terminal; and a light chainvariable region CDR3 of 9 amino acids comprising a Gln, Leu or Trp inthe 1st position from the N-terminal, a Gln in the 2nd position from theN-terminal, a Pro in the 7th position from the N-terminal and a Thr inthe 9th position from the N-terminal.

Another aspect of the present application is directed to a method ofinhibiting cell death in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of theanti-ceramide antibody or an anti-ceramide antibody fragment of thepresent application.

Yet another aspect of the present application is directed to a method oftreating Radiation GI Syndrome or ameliorating a symptom of Radiation GISyndrome in a subject, comprising administering to the subject atherapeutically effective amount of the anti-ceramide antibody or ananti-ceramide antibody fragment of the present application.

Still another aspect of the present application relates to a method forthe mitigation of cell death in GI syndrome in a subject in needthereof. The method comprises administration of an effective amount ofan anti-ceramide antibody, or antigen binding fragment thereof, afterexposure of said subject to penetrating radiation.

Still another aspect of the present invention relates to a method forthe inhibition of apoptosis in GvHD in a subject in need thereof. Themethod comprises administration of an effective amount of ananti-ceramide antibody, or antigen binding fragment thereof, before saidsubject receives a transplant or after said subject receives atransplant, but before the onset of GvHD. In another aspect of thepresent invention, said transplant is a bone marrow transplant.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate one or more embodiments of thepresent disclosure and, together with the written description, serve toexplain the principles of exemplary embodiments of the presentdisclosure.

FIGS. 1A-B show: (A) the variable heavy (VH) and light chain (VL)sequences of mAb 2A2, and (B) amino acid sequence alignment of 2A2 cloneand human germline sequence.

FIGS. 2A-B show: (A) humanized 2A2 heavy chain sequence, and (B)humanized 2A2 heavy chain sequence.

FIG. 3 is an illustration of a biological activity for h2A2, 9H10, 9H11,7B10, 9A2, 6B5, and 6C8 in vitro using crude supernatant.

FIG. 4 is an illustration of an exemplary inhibition with purifiedantibody of Jurkat cell apoptosis, 10 Gy.

FIG. 5 is an illustration of an exemplary inhibition of crypt lethalityusing humanized 2a2 and murine 7B10, 6c8 and 6b5.

FIG. 6 is an illustration of exemplary sequences of mouse antibodies6B5, 6C8, 7B10, 9H10, 9H11, 2A2 and humanized 2A2 antibody (h2A2), aswell as depiction of an anti-ceramide consensus sequence. The alignmentand consensus sequence are generated using the computer program MUSCLEmultiple sequence alignment. Conservation is visualised on the alignmentor a sequence group as a histogram giving the score for each column.Conserved columns are indicated by “*” (score of 11 with default aminoacid property grouping), and columns with mutations where all propertiesare conserved are marked with a “+” (score of 10, indicating allproperties are conserved). “−” means a gap.

FIG. 7 is an illustration of an exemplary 6B5 scFv inhibition of Jurkatcell apoptosis in vitro.

FIG. 8 is an illustration of an exemplary 6B5 scFv protection against GIcrypt depletion in vivo when administered prior to exposure.

FIG. 9 is an illustration of an exemplary 6B5 scFv mitigation against GIcrypt depletion in vivo when administered after exposure.

FIG. 10 is an illustration that anti-ceramide scFv protects intestinalcrypts in a dose-dependent manner. C57BL/6 mice were administeredhumanized anti-ceramide 2A2 (0-1000 micrograms/mouse) or recombinantanti-ceramide scFv 6B5 (0-100 micrograms per mouse) via intravenousinjection 15 min prior to 15 Gy total-body irradiation (TBI). Mice wereeuthanized 3.5 days following TBI, and a section of proximal jejunum wasremoved, cut into 3 15 mm segments, and placed in 4% paraformaldehyde.Proximal jejunum segments were cross-sectioned, mounted and slides wereH&E stained prior to quantification of surviving crypts according to themethod of Withers and Elkind (1970). As the intestinal crypt is the siteof the intestinal stem cell, the microcolony assay is commonly used as asurrogate for intestinal stem cell survival. N=5 mice per group.

FIG. 11 is an illustration that anti-ceramide scFv retains efficacy whenadministered via alternative injections. C57BL/6 mice were administeredhumanized 2A2 anti-ceramide antibody (50 mg/kg) via intravenous (IV),intraperitoneal (IP) or subcutaneous (SC) injection 15 min prior toexposure to 15 Gy total-body irradiation. Alternatively, mice wereadministered 7.5 mg/kg anti-ceramide scFv 6B5 via IV, IP, SC orintramuscular (IM) injection 15 min prior to 15 Gy total-bodyirradiation (TBI). Mice were euthanized 3.5 days following TBI, and asection of proximal jejunum was removed, cut into 3 15 mm segments, andplaced in 4% paraformaldehyde. Proximal jejunum segments werecross-sectioned, mounted and slides were H&E stained prior toquantification of surviving crypts according to the method of Withersand Elkind (1970). As the intestinal crypt is the site of the intestinalstem cell, the microcolony assay is commonly used as a surrogate forintestinal stem cell survival. N=5 mice per group.

FIG. 12 is an illustration that anti-ceramide h2a2 and scFv protect andmitigate the lethal effects of Radiation GI Syndrome. (left panel)C57BL/6 mice were administered humanized anti-ceramide 2A2 (1000micrograms/mouse) via intravenous injection or recombinant anti-ceramidescFv 6B5 (100 micrograms per mouse) via subcutaneous injection 15 minprior to 14.5 Gy total-body irradiation (TBI). Mice received 5×106autologous bone marrow cells 16 hours post TBI and were monitored dailyfor morbidity and mortality. Mice considered moribund were euthanizedimmediately. Data represents a Kaplan-Meier survival plot analyzed byLog-rank test. P<0.05 for both h2A2 and scFv groups vs. saline control.(right panel). Experiment was performed exactly as in left panel, excepthumanized anti-ceramide 2A2 (1000 micrograms/mouse) via intravenousinjection or recombinant anti-ceramide scFv 6B5 (100 micrograms permouse) via subcutaneous injection were administered 24 hours post 14.5Gy total-body irradiation (TBI). N=5 mice per group. P<0.05 for bothh2A2 and scFv groups vs. saline control.

FIG. 13 is an illustration that anti-ceramide scFv protects mice fromlethal acute graft-versus-host disease. C57BL/6 mice (MHC H2^(b)haplotype) were administered PBS or 7.5 mg/kg anti-ceramide scFv 6B5 viaintravenous injection 15 min prior to 1100 cGy split-dose total-bodyirradiation (TBI). Mice received an allogeneic bone marrowtransplantation 16-20 hours post TBI consisting of 5×10⁶ bone marrow(BM) or BM and 2×10⁶ CD5+ naïve T cells from B10.BR donor mice (MHC H2′haplotype). Mice received PBS or 7.5 mg/kg anti-ceramide scFv 6B5 ondays 4, 8, 12 and 16. Mice were scored weekly for GvHD-associatedmorbidity, including weight loss, skin lesions, fur ruffling and lossand kyphosis, and monitored for survival. Data represents a Kaplan-Meiersurvival plot analyzed by Log-rank test. P<0.05 for scFv group vs.saline control.

FIG. 14 is an illustration that anti-ceramide scFv protects mouseintestinal stem cells during lethal acute graft-versus-host disease.C57BL/6 mice (MHC H2^(b) haplotype) were administered PBS, 50 mg/kghumanized h2A2 antibody or 7.5 mg/kg anti-ceramide scFv 6B5 viaintravenous injection 15 min prior to 1100 cGy split-dose total-bodyirradiation (TBI). Mice received an allogeneic bone marrowtransplantation 16-20 hours post TBI consisting of 5×106 bone marrow(BM) or BM and 2×10⁶ CD5+ naïve T cells from B10.BR donor mice (MHCH2^(k2) haplotype). Mice received PBS or 7.5 mg/kg anti-ceramide scFv6B5 on days 4 and 8. Mice were euthanized day 10 post transplant, and asection of proximal jejunum was removed, cut into 3 15 mm segments, andplaced in 4% paraformaldehyde. Proximal jejunum segments werecross-sectioned, mounted and slides were H&E stained prior toquantification of surviving crypts according to the method of Withersand Elkind (1970). As the intestinal crypt is the site of the intestinalstem cell, the microcolony assay is commonly used as a surrogate forintestinal stem cell survival. N=5 mice per group.

FIG. 15 is an illustration that anti-ceramide h2A2 and scFv increaseretention of CD4+ and CD8+ lymphocytes within the mesentery lymph nodes.C57BL/6 mice (MHC H2^(b) haplotype) were administered PBS, 50 mg/kghumanized h2A2 antibody or 7.5 mg/kg anti-ceramide scFv 6B5 viaintravenous injection 15 min prior to 1100 cGy split-dose total-bodyirradiation (TBI). Mice received an allogeneic bone marrowtransplantation 16-20 hours post TBI consisting of 5×10⁶ bone marrow (BMonly) or BM and 2×10⁶ CD5+ naïve T cells from B10.BR donor mice (MHCH2k2 haplotype). Mice subsequently received PBS, h2A2 or scFv 6B5 ondays 4 and 8. Mice were euthanized day 10 post transplant, and mesenterylymph nodes wee analyzed by flow cytometry. Data represents the totalnumber of CD4+ and CD8+ cells from the total donor (H2^(k2) haplotypepositive) CD45+ lymphocyte pool. N=5 mice per group.

DETAILED DESCRIPTION

The following detailed description is presented to enable any personskilled in the art to make and use the invention. For purposes ofexplanation, specific nomenclature is set forth to provide a thoroughunderstanding of the present invention. However, it will be apparent toone skilled in the art that these specific details are not required topractice the invention. Descriptions of specific applications areprovided only as representative examples. The present invention is notintended to be limited to the embodiments shown, but is to be accordedthe widest possible scope consistent with the principles and featuresdisclosed herein.

A “therapeutically effective amount,” as used herein, refers to anamount of a compound is an amount that achieves the desired biologic ortherapeutic effect, namely an amount that prevents, reduces orameliorates one or more symptoms of the enumerated diseases beingtreated or prevented.

The terms “treat,” “treating” or “treatment” as used herein, refers to amethod of alleviating or abrogating a disorder and/or its attendantsymptoms. The terms “prevent,” “preventing” or “prevention,” as usedherein, refer to a method of barring a subject from acquiring a disorderand/or its attendant symptoms. In certain embodiments, the terms“prevent,” “preventing” or “prevention” refer to a method of reducingthe risk of acquiring a disorder and/or its attendant symptoms.

As used herein, the terms “mitigate,” “mitigation” and “mitigating,” inregard to a treatment, refer to the treatment of an acute event afterthe occurrence of said event, for example, mitigating radiation damage24 hours post exposure.

Similarly, as used herein, the terms “protect,” “protection” and“protecting,” in regard to a treatment, refer to the prophylacticadministration of a therapeutic agent for the prevention or inhibitionof an event prior to the occurrence of said event.

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. The term “antibody” is used in thebroadest sense and specifically covers monoclonal antibodies (includingfull length monoclonal antibodies), polyclonal antibodies, multispecificantibodies (e.g., bispecific antibodies), recombinant antibodies, suchas scFv, and antibody fragments so long as they exhibit the desiredbiological activity. By “specifically bind” or “immunoreacts with” ismeant that the antibody reacts with one or more antigenic determinantsof the desired antigen and does not react (i.e., bind) with otherantigens, including polypeptides and lipids or binds at much loweraffinity with other antigens.

The term “antibody” also includes antibody fragments that comprise aportion of a full length antibody, generally the antigen binding orvariable region thereof. Examples of antibody fragments include Fab,Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies;single-chain variable fragment (scFv); and multispecific antibodiesformed from antibody fragments. In certain embodiments of the invention,an antibody fragment, rather than an intact antibody, is used toincrease tissue penetration or tumor penetration. In other embodiments,antibody fragment are further modified to increase its serum half-life.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity.

“Humanized” forms of non-human antibodies are chimeric antibodies whichcontain minimal sequence derived from non-human immunoglobulin. For themost part, humanized antibodies are human immunoglobulins (recipientantibody) in which residues from a hypervariable region of the recipientare replaced by residues from a hypervariable region of a non-humanspecies (donor antibody) such as mouse, rat, rabbit or nonhuman primatehaving the desired specificity, affinity, and/or capacity. Methods formaking humanized and other chimeric antibodies are known in the art.

“Bispecific antibodies” are antibodies that have binding specificitiesfor at least two different antigens. Methods for making bispecificantibodies are known in the art.

The use of “heteroconjugate antibodies” is also within the scope of thepresent invention. Heteroconjugate antibodies are composed of twocovalently joined antibodies. Such antibodies have, for example, beenproposed to target immune system cells to unwanted cells (U.S. Pat. No.4,676,980). It is contemplated that the antibodies can be prepared invitro using known methods in synthetic protein chemistry, includingthose involving crosslinking agents.

As used herein, the term “LD₅₀” refers to “Lethal Dose, 50%” or “medianlethal dose” and is the amount of a substance required to kill 50% of atest population.

Extracellular Ceramide is Required for Radiation-Induced Apoptosis

Lipid rafts, which are distinct plasma membrane microdomains comprisedof cholesterol tightly associated with sphingolipids, in particularsphingomyelin, creating a liquid-ordered domain within theliquid-disordered bulk plasma membrane. Rafts differ in their proteinand lipid composition from the surrounding membrane, housing signalingmolecules including multiple glycosylphosphatidylinositol (GPI)-anchoredproteins, doubly-acylated tyrosine kinases of the Src family andtransmembrane proteins. In addition, rafts serve as sites that multiplereceptors translocate into or out of upon their activation, includingthe B cell receptor (BCR) upon encountering antigen. Evidence suggeststhat these translocation events are crucial for multiple signaltransduction cascades.

Sphingolipids are structural components of cell membranes and importantregulators of signal transduction through the generation of ceramide.C16 ceramide has important roles in differentiation, proliferation andgrowth arrest. It is also an essential component of apoptotic signaling.Ceramide generation has been identified as requisite for multiplecytokine-, virus/pathogen-, environmental stress-, andchemotherapeutic-induced apoptotic events. In addition, Ceramide-richregions on the plasma membrane of target cells are critical forsensitivity to cytotoxic T lymphocyte (CTL)-induced cell death.

Treatment and Prevention of the Lethal GI Syndrome

Cycling crypt base columnar (CBC) cells located at positions 1-3 fromthe bottom of the crypt of Lieberkuhn, have recently been defined as apopulation of intestinal stem cells. This group of cells proliferatesand differentiates incessantly, replenishing the physiologic loss ofenterocytes and other differentiated epithelial cells from the villusapex, thus maintaining the anatomical and functional integrity of themucosa. A complete or near-complete depletion of this compartmentappears required to permanently destroy the crypt-villus unit, whilesurviving stem cell clonogens, albeit even one per crypt, are capable ofregenerating a fully functional crypt.

Radiation targets both the gastrointestinal microvasculature andintestinal stem cell compartments. Dysfunction of the microvascularendothelium, detected as apoptosis at four hours following radiation,represents a principle lesion leading to the GI syndrome. Endothelialdysfunction converts lesions to CBCs from sublethal to lethal, resultingin loss of regenerative crypts and promoting GI toxicity.Immunohistochemical and labeling studies with [³H]TdR and BrdUrdrevealed that crypt stem cell death does not occur acutely afterradiation exposure. Rather, the earliest detectable response is atemporary dose-dependent delay in progression through a late S-phasecheckpoint and mitotic arrest, apparently signaled by radiation-inducedDNA double strand breaks (dsb). A rapid apoptotic death occurs in growtharrested cells during the first 24 hours post irradiation that, at 12Gy, equals 33% of the total death. In mammalian cells, DNA dsbs activatepathways of DNA damage recognition and repair, and a coordinatedregulation of cell cycle checkpoint activity. The intestinal stem cellmitotic arrest appears to represent a regulated event in this pathway. Amitotic form of death occurs during this second 24 hours, representing66% of overall death. No significant change in crypt number perintestinal circumference is apparent at this stage although crypt sizeprogressively decreases due to continued normal migration of crypttransit and differentiated cells from the crypt into the epitheliallining of the villus and loss from the villus tip. Resumption of mitoticactivity at 12-18 hours is associated with a rapid depletion of cryptstem cell clonogens and reduction in crypt number per circumference.

The lethality of GI stem cell clonogens is best assessed by the numberof crypts surviving at 3.5 days after radiation exposure, whichdecreases exponentially as the dose increases (C. S. Potten and M.Loeffler, Development 110 (4), 1001 (1990), H. R. Withers, Cancer 28(1), 75 (1971), and J. G. Maj, F. Paris, A. Haimovitz-Friedman et al.,Cancer Res 63, 4338 (2003)). Crypts that contain surviving stem cellsproliferate at an accelerated rate, producing typical regenerativecrypts that split or bud to generate new crypts, until the intestinalmucosa regains a normal architecture. TBI experiments in several mousemodels have demonstrated that the number of surviving crypt stem cellsafter exposure to 8-12 Gy is usually sufficient to support a completerecovery of the mucosa. At higher doses, however, massive stem cellclonogen loss may lead to a near total collapse of the crypt-villussystem, mucosal denudation and animal death from the GI syndrome. Thethreshold dose for inducing the GI death, and the TBI dose producing 50%GI lethality (LD₅₀), appear to be strain-specific. Autopsy studies ofC57BL/6 mice exposed to TBI revealed that 25% of the mice exposed to 14Gy and 100% of those exposed 15 Gy succumbed to the GI syndrome at6.8+/−0.99 days, predicting an LD₅₀ for GI death between 14 and 15 Gy.In contrast, the reported LD₅₀D6 (the LD₅₀ at day 6, serving as asurrogate marker for GI death) for BALB/c mice is 8.8+/−0.72 Gy,11.7+/−0.22 Gy for BDF1 mice, 12.5+/−0.1 Gy for C3H/He mice, 14.9 Gy(95% confidence limits 13.9-16.0 Gy) for C3H/SPF mice, and 16.4+/−0.2 Gyfor B6CF1 mice, indicating a strain-specific spectrum in mousesensitivity to death from the GI syndrome. Strain variations in thesensitivity of other organs to radiation, such as the bone marrow andlung have also been reported.

Classically, penetrating radiation (IR) was thought to kill cells bydirect damage to genomic DNA, causing genomic instability and resultingin reproductive cell death. Haimovitz-Friedman et al. (Cancer Res 63,4338 (2003)) demonstrated in a nuclei-free system that apoptoticsignaling can alternately be generated by the interaction of IR withcellular membranes. Ceramide mediated raft clustering is involved inIR-induced apoptosis and clonogenic cell death. It has long beenaccepted that the clonogenic compartment of the gastrointestinal (GI)mucosa is the specific and direct target for radiation in inducing GIdamage.

One aspect of the present application is directed to an anti-ceramideantibody, or an antigen-binding fragment thereof, comprising: a heavychain variable region CDR1 of 10 amino acids comprising a Gly in the 1stposition from the N-terminal, a Tyr or Phe in the 2nd position from theN-terminal, a Phe or Leu in the 4th position from the N-terminal, and aThr or His in the 6th position from the N-terminal and a His or Asn inthe 10th position from the N-terminal; a heavy chain variable regionCDR2 of 16-17 amino acids comprising a Asn or Ile in the 2nd positionfrom the N-terminal, a Phe or Ser in the 4th position from theN-terminal, a Thr in the 9th position from the C-terminal, a Tyr in the7th position from the C-terminal, an Asn in the 6th position from theC-terminal, a Lys or Ala in the 2nd and 4th positions from theC-terminal; a heavy chain variable region CDR3 of 7 to 11 amino acidscomprising a Tyr or Thr at the 4th position from the N-terminal; a lightchain variable region CDR1 of 10-16 amino acids comprising an Ala or Serin the 2nd position from the N-terminal, a Ser in the 3rd position fromthe N-terminal, a Ser or Asp in the 5th position from the N-terminal,and a Tyr, Ser or Phe in the 3th position from the C-terminal; a lightchain variable region CDR2 of 7 amino acids comprising a Ser or Asn inthe 3rd position from the N-terminal, a Lys or Ser in the 5th positionfrom the N-terminal and a Ser or Asp in the 7th position from theN-terminal; and a light chain variable region CDR3 of 9 amino acidscomprising a Gln, Leu or Trp in the 1st position from the N-terminal, aGln in the 2nd position from the N-terminal, a Pro in the 7th positionfrom the N-terminal and a Thr in the 9th position from the N-terminal.

As used herein, the term “CDR” refers to the “complementaritydetermining region” of an immunoglobulin (antibody) molecule. CDRs arepart of the variable domain in an antibody where the antibody binds toits specific antigen. CDRs are crucial to the diversity of antigenspecificities generated by lymphocytes. There are three CDR per variabledomain (i.e., CDR1, CDR2 and CDR3 in the variable domain of the lightchain and CDR1, CDR2 and CDR3 in the variable domain of the heavy chain)for a total of 12 CDRs in an IgG molecule and 60 CDRs in an IgMmolecule. Within the variable domain, CDR1 and CDR2 are found in thevariable (V) region of a polypeptide chain, CDR3 shows the greatestvariability as it is encoded by a recombination of the VJ in the case ofa light chain region and VDJ in the case of heavy chain regions.

In some embodiments, the heavy chain variable region CDR1 of theanti-ceramide antibody, or antigen-binding fragment thereof, comprisesthe sequence GYTFTDHTIH (SEQ ID NO: 1), said heavy chain variable regionCDR2 comprises the sequence YNYPRDGSTKYNEKFKG (SEQ ID NO: 2), a heavychain variable region CDR3 comprising the sequence GFITTVVPSAY (SEQ IDNO: 3), said light chain variable region CDR1 comprises the sequenceRASKSISKYLA (SEQ ID NO: 4), a light chain variable region CDR2comprising the sequence SGSTLQS (SEQ ID NO: 5), and said light chainvariable region CDR3 comprising the sequence QQHNEYPWT (SEQ ID NO: 6).

In further embodiments, the anti-ceramide antibody, or antigen-bindingfragment thereof, comprises: a heavy chain variable region sequencecomprising the sequence QVQLQQSDAELVKPGASVKISCKVSGYTFTDHTIHWMKQRPEQGLEWIGYNPRDGSTKYNEKFGKATLTDADKSSSTAYMQLNSLTSEDSAVYFCAKGFITTVVPSAYWGQGTLVTVSA (SEQ ID NO: 7), or a sequence with at least about 80%,85%, 90% or 95% sequence identity to a heavy chain variable regionsequence comprising SEQ ID NO: 7; and/or a light chain variable regionsequence comprising SEQ ID NO: 8, or a sequence with at least about 80%,85%, 90% or 95% sequence identity to a light chain variable regionsequence comprising the sequence DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPW TFGGGTKLEIK(SEQ ID NO: 8).

In other embodiments, the heavy chain variable region CDR1 of theanti-ceramide antibody, or antigen-binding fragment thereof, comprisesthe sequence GYAFSSYWMN (SEQ ID NO: 9), said heavy chain variable regionCDR2 comprises the sequence QIYPGDGDTNYNGKFKG (SEQ ID NO: 10), a heavychain variable region CDR3 comprising the sequence RCYYGLYFDV (SEQ IDNO: 11), said light chain variable region CDR1 comprises the sequenceKASQDINRYLS (SEQ ID NO: 12), a light chain variable region CDR2comprising the sequence RANRLVD (SEQ ID NO: 13), and said light chainvariable region CDR3 comprising the sequence LQYDEFPYT (SEQ ID NO: 14).

In further embodiments, the anti-ceramide antibody, or antigen-bindingfragment thereof, comprises: a heavy chain variable region sequencecomprising the sequenceQVQLQQSGAELVKPGASVKISCKASGYAFSSYWMNWVKQRPGKGLEWIGQIYPGDGDTNYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCTRRCYYGLYFDVWGT GTTVTVSS (SEQID NO: 15), or a sequence with at least about 80%, 85%, 90% or 95%sequence identity to a heavy chain variable region sequence comprisingSEQ ID NO: 15; and/or a light chain variable region sequence comprisingthe sequence DIKMTQSPSSRYASLGERVTITCKASQDINRYLSWFQQKPGKSPKTLIYRANRLVDGVPSSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIK (SEQ ID NO: 16), or a sequence withat least about 80%, 85%, 90% or 95% sequence identity to a light chainvariable region sequence comprising SEQ ID NO: 16.

In yet other embodiments, the heavy chain variable region CDR1 of theanti-ceramide antibody, or antigen-binding fragment thereof, comprisesthe sequence GYTFTSYWMH (SEQ ID NO: 17), said heavy chain variableregion CDR2 comprises the sequence YINPSSGYTKYNQFKD (SEQ ID NO: 18), aheavy chain variable region CDR3 comprising the sequence GGYYGFAY (SEQID NO: 19), said light chain variable region CDR1 comprises the sequenceSASSSVSYMY(SEQ ID NO: 20), a light chain variable region CDR2 comprisingthe sequence LTSNLAS (SEQ ID NO: 21), and said light chain variableregion CDR3 comprising the sequence QQWSSNPLT (SEQ ID NO: 22).

In further embodiments, the anti-ceramide antibody, or antigen-bindingfragment thereof, comprises: a heavy chain variable region sequencecomprising the sequenceQVQLQQSGAELAKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGYINPSSGYTKYNQKFKDKATLTADKSSSTAYMQLSSLTYEDSAVYYCARGGYYGFAYWGQGT LVTVSA (SEQ IDNO: 23), or a sequence with at least about 80%, 85%, 90% or 95% sequenceidentity to a heavy chain variable region sequence comprising SEQ ID NO:23; and/or a light chain variable region sequence comprising thesequence QIVLTQSPALMSASPGEKVTMTCSASSSVSYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO: 24), or a sequence withat least about 80%, 85%, 90% or 95% sequence identity to a light chainvariable region sequence comprising SEQ ID NO: 24.

In still other embodiments, the heavy chain variable region CDR1 of theanti-ceramide antibody, or antigen-binding fragment thereof, comprisesthe sequence GFSLTGYGVH (SEQ ID NO: 25), said heavy chain variableregion CDR2 comprises the sequence VIWSGGSTDYNAAFIS (SEQ ID NO: 26), aheavy chain variable region CDR3 comprising the sequence NYGYDYAMDY (SEQID NO: 27), said light chain variable region CDR1 comprises the sequenceRASQSIGTSIH (SEQ ID NO: 28), a light chain variable region CDR2comprising the sequence YASESIS (SEQ ID NO: 29), and said light chainvariable region CDR3 comprising the sequence QQSNSWPFT (SEQ ID NO: 30).

In further embodiments, the anti-ceramide antibody, or antigen-bindingfragment thereof, comprises: a heavy chain variable region sequencecomprising SEQ ID NO: 31, or a sequence with at least about 80%, 85%,90% or 95% sequence identity to a heavy chain variable region sequencecomprising the sequence QVQLKQSGPGVQPSSLSITCTVSGFSLTSYGVHWVRQSPGKGLEWLGVIWSGGSTDYNAAFISRLSISKDNSKSQVFFKMNSLQADDTAIYYCARNYGYDYAMDYWGQGTSVTVSS (SEQ ID NO: 31); and/or a lightchain variable region sequence comprising the sequenceDILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPFTFGSGTKLEIK (SEQ ID NO: 32), or a sequence with at leastabout 80%, 85%, 90% or 95% sequence identity to a light chain variableregion sequence comprising SEQ ID NO: 32.

In even other embodiments, the heavy chain variable region CDR1 of theanti-ceramide antibody, or antigen-binding fragment thereof, comprisesthe sequence GYTFTNYWMH (SEQ ID NO: 33), said heavy chain variableregion CDR2 comprises the sequence AIYPGDSDTSYNQKFKG (SEQ ID NO: 34), aheavy chain variable region CDR3 comprising the sequence GLYYGYD (SEQ IDNO: 35), said light chain variable region CDR1 comprises the sequenceKSSQSLIDSDGKTFLN (SEQ ID NO: 36), a light chain variable region CDR2comprising the sequence LVSKLDS (SEQ ID NO: 37), and said light chainvariable region CDR3 comprising the sequence WQGTHFPYT (SEQ ID NO: 38).

In further embodiments, the anti-ceramide antibody, or antigen-bindingfragment thereof, comprises: a heavy chain variable region sequencecomprising the sequence EVQLQQSGTVLARPGASVKMSCKASGYTFTNYWMHWVKQRPVQGLEWIGAIYPGDSDTSYNQKFKGKAKLTAVTSTSTAFMELSSLTNED SAVYYCTGLYYGYD WGQGTTLTVSS(SEQ ID NO: 39), or a sequence with at least about 80%, 85%, 90% or 95%sequence identity to a heavy chain variable region sequence comprisingSEQ ID NO: 39; and/or a light chain variable region sequence comprisingSEQ ID NO: 40, or a sequence with at least about 80%, 85%, 90% or 95%sequence identity to a light chain variable region sequence comprisingthe sequence DVLMTQTPLTLSVTIGQPASISCKSSQSLIDSDGKTFLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGLYYCWQGTHFPYTFGGGTKLEIK (SEQ ID NO: 40).

In still yet other embodiments, the anti-ceramide antibody, orantigen-binding fragment thereof, comprises: a) a heavy chain variableregion sequence comprising a sequence selected from the group consistingof SEQ ID NOs: 7, 15, 23, 31 and 39, or a sequence with at least about80%, 85%, 90% or 95% sequence identity to a heavy chain variable regionsequence comprising a sequence selected from the group consisting of SEQID NOs: 7, 15, 23, 31 and 39; and/or a) a light chain variable regionsequence comprising a sequence selected from the group consisting of SEQID NOs: 8, 16, 24, 32 and 40, or a sequence with at least about 80%,85%, 90% or 95% sequence identity to a light chain variable regionsequence comprising a sequence selected from the group consisting of SEQID NOs: 8, 16, 24, 32 and 40.

In particular embodiments, the anti-ceramide antibody is selected fromthe group consisting of monoclonal antibody, chimeric antibody,humanized antibody, human antibody, recombinant antibody and scFv.

Another aspect of the present application is directed to ananti-ceramide single-chain variable fragment (scFv) that binds to thesame antigenic determinant as the anti-ceramide antibody of the presentapplication. The scFv comprises: a heavy chain variable region CDR1 of10 amino acids comprising a Gly in the 1st position from the N-terminal,a Tyr or Phe in the 2nd position from the N-terminal, a Phe or Leu inthe 4th position from the N-terminal, and a Thr or His in the 6thposition from the N-terminal and a His or Asn in the 10th position fromthe N-terminal; a heavy chain variable region CDR2 of 16-17 amino acidscomprising a Asn or Ile in the 2nd position from the N-terminal, a Pheor Ser in the 4th position from the N-terminal, a Thr in the 9thposition from the C-terminal, a Tyr in the 7th position from theC-terminal, an Asn in the 6th position from the C-terminal, a Lys or Alain the 2nd and 4th positions from the C-terminal; a heavy chain variableregion CDR3 of 7 to 11 amino acids comprising a Tyr or Thr at the 4thposition from the N-terminal; a light chain variable region CDR1 of10-16 amino acids comprising an Ala or Ser in the 2nd position from theN-terminal, a Ser in the 3rd position from the N-terminal, a Ser or Aspin the 5th position from the N-terminal, and a Tyr, Ser or Phe in the3th position from the C-terminal; a light chain variable region CDR2 of7 amino acids comprising a Ser or Asn in the 3rd position from theN-terminal, a Lys or Ser in the 5th position from the N-terminal and aSer or Asp in the 7th position from the N-terminal; and a light chainvariable region CDR3 of 9 amino acids comprising a Gln, Leu or Trp inthe 1st position from the N-terminal, a Gln in the 2nd position from theN-terminal, a Pro in the 7th position from the N-terminal and a Thr inthe 9th position from the N-terminal.

A single-chain variable fragment (scFv) is not actually a fragment of anantibody, but instead is a fusion protein of the variable regions of theheavy (VH) and light chains (VL) of immunoglobulins, connected with ashort linker peptide of ten to about 25 amino acids. The linker isusually rich in glycine for flexibility, as well as serine or threoninefor solubility, and can either connect the N-terminus of the VH with theC-terminus of the VL, or vice versa. The scFv retains the specificity ofthe original immunoglobulin, despite removal of the constant regions andthe introduction of the linker.

In some embodiments, the anti-ceramide antibodies, antigen-bindingfragments thereof, or scFv are produced using recombinant DNAtechnologies. Procedures for the expression and purification ofrecombinant proteins are well established in the art.

In order to express the anti-ceramide antibody, antigen-binding fragmentthereof, or scFv of the present application in a biological system, apolynucleotide that encodes the anti-ceramide antibody, antigen-bindingfragment thereof, or scFv is constructed. In certain embodiments, therecombinant polynucleotide is codon optimized for expression in aselected prokaryotic or eukaryotic host cell, such as a bacterial,mammalian, plant or insect cell. To facilitate replication andexpression, the polynucleotide can be incorporated into a vector, suchas a prokaryotic or a eukaryotic expression vector. Although thepolynucleotide disclosed herein can be included in any one of a varietyof vectors (including, for example, bacterial plasmids; phage DNA;baculovirus; yeast plasmids; vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, pseudorabies, adenovirus, adeno-associated virus, retrovirusesand many others), most commonly the vector will be an expression vectorsuitable for generating polypeptide expression products. In anexpression vector, the polynucleotide encoding the anti-ceramideantibody, antigen-binding fragment thereof, or scFv is typicallyarranged in proximity and orientation to an appropriate transcriptioncontrol sequence (promoter, and optionally, one or more enhancers) todirect mRNA synthesis. That is, the polynucleotide sequence of interestis operably linked to an appropriate transcription control sequence.Examples of such promoters include: the immediate early promoter of CMV,LTR or SV40 promoter, polyhedron promoter of baculovirus, E. coli lac ortrp promoter, phage T7 and lambda P_(L) promoter, and other promotersknown to control expression of genes in prokaryotic or eukaryotic cellsor their viruses. The expression vector typically also contains aribosome binding site for translation initiation, and a transcriptionterminator. The vector optionally includes appropriate sequences foramplifying expression. In addition, the expression vectors optionallycomprise one or more selectable marker genes to provide a phenotypictrait for selection of transformed host cells, such as dihydrofolatereductase or neomycin resistance for eukaryotic cell culture, or such astetracycline or ampicillin resistance in E. coli.

The expression vector can also include additional expression elements,for example, to improve the efficiency of translation. These signals caninclude, e.g., an ATG initiation codon and adjacent sequences. In somecases, for example, a translation initiation codon and associatedsequence elements are inserted into the appropriate expression vectorsimultaneously with the polynucleotide sequence of interest (e.g., anative start codon). In such cases, additional translational controlsignals are not required. However, in cases where only a polypeptidecoding sequence, or a portion thereof, is inserted, exogenoustranslational control signals, including an ATG initiation codon isprovided for expression of the anti-ceramide antibody or scFv. Theinitiation codon is placed in the correct reading frame to ensuretranslation of the polynucleotide sequence of interest. Exogenoustranscriptional elements and initiation codons can be of variousorigins, both natural and synthetic. If desired, the efficiency ofexpression can be further increased by the inclusion of enhancersappropriate to the cell system in use.

Expression vectors carrying the anti-ceramide antibody or scFv of thepresent application can be introduced into host cells by any of avariety of well-known procedures, such as electroporation, liposomemediated transfection, calcium phosphate precipitation, infection,transfection and the like, depending on the selection of vectors andhost cells.

Host cells that contain anti-ceramide antibody, antigen-binding fragmentthereof, or scFv-encoding nucleic acids are, thus, also a feature ofthis disclosure. Favorable host cells include prokaryotic (i.e.,bacterial) host cells, such as E. coli, as well as numerous eukaryotichost cells, including fungal (e.g., yeast, such as Saccharomycescerevisiae and Picchia pastoris) cells, insect cells, plant cells, andmammalian cells (such as CHO cells).

The host cells can be cultured in conventional nutrient media modifiedas appropriate for activating promoters, selecting transformants, oramplifying the inserted polynucleotide sequences. The cultureconditions, such as temperature, pH and the like, are typically thosepreviously used with the host cell selected for expression, and will beapparent to those skilled in the art. A host cell is optionally chosenfor its ability to modulate the expression of the inserted sequences orto process the expressed protein in the desired fashion. Suchmodifications of the protein include, but are not limited to,glycosylation, (as well as, e.g., acetylation, carboxylation,phosphorylation, lipidation and acylation). Post-translationalprocessing for example, which cleaves a precursor form into a matureform of the protein (for example, by a furin protease) is optionallyperformed in the context of the host cell. Different host cells such as3T3, COS, CHO, HeLa, BHK, MDCK, 293, WI38, etc. have specific cellularmachinery and characteristic mechanisms for such post-translationalactivities and can be chosen to ensure the correct modification andprocessing of the introduced, foreign protein.

For long-term, high-yield production of recombinant anti-ceramideantibody, antigen-binding fragment thereof, or scFv polypeptide, stableexpression systems are typically used. For example, polynucleotidesencoding an anti-ceramide antibody, antigen-binding fragment thereof, orscFv are introduced into the host cell using expression vectors whichcontain viral origins of replication or endogenous expression elementsand a selectable marker gene. Following the introduction of the vector,cells are allowed to grow for 1-2 days in an enriched media before theyare switched to selective media. The purpose of the selectable marker isto confer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.For example, resistant groups or colonies of stably transformed cellscan be proliferated using tissue culture techniques appropriate to thecell type. Host cells transformed with a nucleic acid encoding ananti-ceramide antibody, antigen-binding fragment thereof, or scFv areoptionally cultured under conditions suitable for the expression andrecovery of the encoded protein from cell culture.

Following transduction of a suitable host cell line and growth of thehost cells to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period. The secretedpolypeptide product is then recovered from the culture medium.Alternatively, cells can be harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification. Eukaryotic or microbial cells employed inexpression of proteins can be disrupted by any convenient method,including freeze-thaw cycling, sonication, mechanical disruption, or useof cell lysing agents, or other methods, which are well know to thoseskilled in the art.

Expressed anti-ceramide antibody, antigen-binding fragment thereof, orscFv can be recovered and purified from recombinant cell cultures by anyof a number of methods well known in the art, including ammonium sulfateor ethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography (e.g., using any of the taggingsystems noted herein), hydroxylapatite chromatography, and lectinchromatography. Protein refolding steps can be used, as desired, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed in the finalpurification steps.

In certain examples, the nucleic acids are introduced into vectorssuitable for introduction and expression in prokaryotic cells, e.g., E.coli cells. In some embodiments, the expression vector is introduced(e.g., by electroporation) into a suitable bacterial host. In anotherexample, a polynucleotide sequence that encodes an anti-ceramideantibody or scFv is introduced into insect cells using a BaculovirusExpression Vector System (BEVS). Similarly, alternative insect cells canbe employed, such as SF21 which is closely related to the SF9, and theHigh Five (Hi5) cell line derived from a cabbage looper, Trichoplusiani.

In yet other embodiments, the anti-ceramide antibody, antigen-bindingfragment thereof, or scFv is expressed in vivo by a plasmid vector or aviral vector.

In certain embodiments, the anti-ceramide antibodies, antigen-bindingfragments thereof, and scFv are produced by chemical synthesis. Briefly,an anti-ceramide antibody, antigen-binding fragment thereof, or scFv maybe synthesized by coupling the carboxyl group or C-terminus of one aminoacid to the amino group or N-terminus of another. Due to the possibilityof unintended reactions, protecting groups may be necessary. Chemicalpeptide synthesis starts at the C-terminal end of the peptide and endsat the N-terminus. This is the opposite of protein biosynthesis, whichstarts at the N-terminal end.

In some embodiments, the anti-ceramide antibodies, antigen-bindingfragments thereof, and scFv may be synthesized using traditional liquid-or solid-phase synthesis. Fmoc and t-Boc solid phase peptide synthesis(SPPS) can be employed to grow the peptides from carboxy toamino-terminus. In certain embodiments, the last “amino acid” added tothe reaction is PEGylated. This last amino acid is often referred to asa carboxyl-PEG-amine, carboxyl-PEO-amine, or amine-PEG-acid, whereby theamine is blocked to protect against reaction and the acid is free toreact with the amine group from the previously added amino acid in thereaction. PEG (polyethylene glycol) and PEO (polyethylene oxide) arepolymers composed of repeating subunits of ethylene glycol and ethyleneoxide monomers. In one embodiment, a PEGylated anti-ceramide antibody,antigen-binding fragments thereof, and scFv would have the PEG moietyconnected to the histidine residue (H) at the amino-terminus of thepolypeptide. In one embodiment, the PEG moiety is 5 to 30 kDa in size.In another embodiment, the PEG moiety is 10 to 20 kDa in size.

In addition to using PEGylated end amino acid during synthesis, ananti-ceramide antibody, antigen-binding fragment thereof, or scFv may bePEGylated by PEGylation. PEGylation is the process of covalentattachment of polyethylene glycol polymer chains to another molecule,normally a drug or therapeutic protein. PEGylation can be achieved byincubation of a reactive derivative of PEG with the target anti-ceramideantibody or scFv. The covalent attachment of PEG to an anti-ceramideantibody or scFv can “mask” the anti-ceramide antibody, antigen-bindingfragment thereof, or scFv from the host's immune system (reducedimmunogenicity and antigenicity), increase the hydrodynamic size (sizein solution) of the anti-ceramide antibody, antigen-binding fragmentthereof, or scFv which prolongs its circulatory time by reducing renalclearance. PEGylation can also provide water solubility to hydrophobicproteins.

Method of Inhibiting Apoptosis

Still another aspect of the present application is directed to a methodof inhibiting cell death in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of ananti-ceramide antibody, or antigen-binding fragment thereof, of thepresent application. In some embodiments, the anti-ceramide antibody isan anti-ceramide scFv.

In some embodiments, the cell death is associated with a diseaseselected from the group consisting of graft versus host disease,radiation disease, GI syndrome and autoimmune disease. In some furtherembodiments, the disease is radiation disease or GI syndrome and theanti-ceramide antibody, or antigen-binding fragment thereof, isadministered before the subject is exposed to radiation.

Another aspect of the present application is directed to a method forthe mitigation of cell death in GI syndrome in a subject in needthereof. The method comprises the administration of an effective amountof an anti-ceramide antibody. In some embodiments, the method comprisesadministering said anti-ceramide antibody to said subject immediatelyafter exposure of said subject to penetrating radiation. In otherembodiments, the method comprises administering said anti-ceramideantibody to said subject within one hour after exposure of said subjectto penetrating radiation. In still other embodiments, the methodcomprises administering said anti-ceramide antibody to said subjectwithin 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 18 hours after exposure ofsaid subject to penetrating radiation. In a particular embodiment, themethod comprises administering said anti-ceramide antibody to saidsubject within 24 hours after exposure of said subject to penetratingradiation. In other embodiments, the method comprises administering saidanti-ceramide antibody to said subject within 30, 36, 42, 48, 54, 60, 66or 72 hours after exposure of said subject to penetrating radiation. Inother embodiments, the method comprises administering said anti-ceramideantibody to said subject within 48, 36, 24, 18, 12, 10, 8, 6, 4, 2 or 1hour(s), or within 45, 30 or 15 minutes before exposure of said subjectto penetrating radiation.

In other further embodiments, the disease is graft versus host diseaseand the anti-ceramide antibody, or antigen-binding fragment thereof, isadministered before the subject receives a transplant. In someembodiments, the transplant is a bone marrow transplant. In still otherfurther embodiments, the anti-ceramide antibody, or antigen-bindingfragment thereof, is administered after the subject receives atransplant, but before the onset of graft versus host disease. In evenstill other further embodiments, the anti-ceramide antibody, orantigen-binding fragment thereof, is administered to a subject in needthereof after the onset of graft versus host disease in an amounteffective for the mitigation of apoptosis in graft versus host disease.

Antibody Administration

The antibody, or antigen-binding fragment thereof, may be administeredto the subject with known methods, such as intravenous administration asa bolus or by continuous infusion over a period of time, byintramuscular, intraperitoneal, intracerobrospinal, subcutaneous,intra-articular, intrasynovial, intrathecal, oral, topical, orinhalation routes.

Antibodies and antigen-binding fragments thereof of the invention can beadministered in the usually accepted pharmaceutically acceptablecarriers. Acceptable carriers include, but are not limited to, saline,buffered saline, glucose in saline. Solid supports, liposomes,nanoparticles, microparticles, nanospheres or microspheres may also beused as carriers for administration of the antibodies or antigen-bindingfragment thereof.

The appropriate dosage (“therapeutically effective amount”) of theantibody, or antigen-binding fragment thereof, will depend, for example,on the condition to be treated, the severity and course of thecondition, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, the type of antibody, or antigen-bindingfragment thereof, used, and the discretion of the attending physician.The antibody, or antigen-binding fragment thereof, is suitablyadministered to the patent at one time or over a series of treatmentsand may be administered to the patent at any time from diagnosisonwards. The antibody, or antigen-binding fragment thereof, may beadministered as the sole treatment or in conjunction with other drugs ortherapies useful in treating the condition in question.

As a general proposition, the therapeutically effective amount of theantibody, or antigen-binding fragment thereof, administered will be inthe range of about 1 ng/kg body weight/day to about 100 mg/kg bodyweight/day whether by one or more administrations. In a particularembodiments, the range of antibody administered is from about 1 ng/kgbody weight/day to about 1 μg/kg body weight/day, 1 ng/kg bodyweight/day to about 100 ng/kg body weight/day, 1 ng/kg body weight/dayto about 10 ng/kg body weight/day, 10 ng/kg body weight/day to about 1μg/kg body weight/day, 10 ng/kg body weight/day to about 100 ng/kg bodyweight/day, 100 ng/kg body weight/day to about 1 μg/kg body weight/day,100 ng/kg body weight/day to about 10 pig/kg body weight/day, 1 μg/kgbody weight/day to about 10 pig/kg body weight/day, 1 μg/kg bodyweight/day to about 100 pig/kg body weight/day, 10 pig/kg bodyweight/day to about 100 pig/kg body weight/day, 10 pig/kg bodyweight/day to about 1 mg/kg body weight/day, 100 μg/kg body weight/dayto about 10 mg/kg body weight/day, 1 mg/kg body weight/day to about 100mg/kg body weight/day and 10 mg/kg body weight/day to about 100 mg/kgbody weight/day.

In another embodiment, the antibody, or antigen-binding fragmentthereof, is administered at a dosage range of 1 ng-10 ng per injection,10 ng to 100 ng per injection, 100 ng to 1 μg per injection, 1 μg to 10μg per injection, 10 μg to 100 μg per injection, 100 μg to 1 mg perinjection, 1 mg to 10 mg per injection, 10 mg to 100 mg per injection,and 100 mg to 1000 mg per injection.

In another particular embodiment, the dose range of antibody, orantigen-binding fragment thereof, administered is from about 1 ng/kg toabout 100 mg/kg In still another particular embodiment, the range ofantibody administered is from about 1 ng/kg to about 10 ng/kg, about 10ng/kg to about 100 ng/kg, about 100 ng/kg to about 1 μg/kg, about 1μg/kg to about 10 μg/kg, about 10 μg/kg to about 100 μg/kg, about 100μg/kg to about 1 mg/kg, about 1 mg/kg to about 10 mg/kg, about 10 mg/kgto about 100 mg/kg, about 0.5 mg/kg to about 30 mg/kg, and about 1 mg/kgto about 15 mg/kg.

In other particular embodiments, the amount of antibody, orantigen-binding fragment thereof, administered is, or is about, 0.0006,0.001, 0.003, 0.006, 0.01, 0.03, 0.06, 0.1, 0.3, 0.6, 1, 3, 6, 10, 30,60, 100, 300, 600 and 1000 mg/day. As expected, the dosage will bedependent on the condition, size, age and condition of the patient.

The antibody, or antigen-binding fragment thereof, may be administered,as appropriate or indicated, a single dose as a bolus or by continuousinfusion, or as multiple doses by bolus or by continuous infusion.Multiple doses may be administered, for example, multiple times per day,once daily, every 2, 3, 4, 5, 6 or 7 days, weekly, every 2, 3, 4, 5 or 6weeks or monthly. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniques.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Figures and Tables, are incorporatedherein by reference.

Example 1: 2A2 Ab Humanization and Production

Cloning of Variable Light and Heavy Chain of 2A2 from Hybridoma

2A2 hybridoma cells were harvested by centrifugation and total RNA wasextracted from cells using RNA purification kit. This total RNA was usedfor cDNA synthesis and finally V-region genes of 2A2 were isolated usingprimer sets published in “Phage display manual”. FIG. 1A shows thevariable heavy (VH) and light chain sequences (VL) of 2A2.

Humanization of 2A2 Variable Region

Usually, rodent antibodies can be immunogenic to human and cause veryserious side effects including the HAMA (human anti-mouse antibodies)response or anaphylactic shock. To overcome this problem, antibodyengineering has been used to humanize non-human antibodies. Therefore,the CDR grafting method was used to humanize the VL and VH of 2A2.

CDR grafting is currently the most frequently used strategy for thehumanization of rodent mAbs. In this approach, CDR loops that make upthe antigen-binding site of the rodent mAb are grafted into thecorresponding human framework regions.

To identify human VL and VH homologous to those of 2A2, the variableregions of 2A2 were compared with variable regions of human germlinesequences using the VBASE online database (vbase.mrc-cpe.cam.ac.uk). Asa result, two human germline VL and VH sequences were found. Amino acidsequence alignment of 2A2 clones and human germline sequence is shown inFIG. 1B.

The selected 2A2 VH sequence was found to be most homologous to thehuman V gene 1-46 from the VH1 family and human J gene JH6. The selected2A2 VL sequence was found to be most homologous with the human V gene A1from the Vk2 family and human J gene Jk2. So, synthesized VL and VH thateach contained three of mouse CDR sequences were grafted into theselected human framework sequences for humanization of 2A2 mAb.

Vector Construction for the Expression of Humanized 2A2 IgG1 inMammalian Cells

2A2 mAb is originally murine IgM. IgM antibodies are converted to theIgG1 format because IgG1 is the most abundant in serum (9 mg/ml), itshalf-life (21 days) is longer than any other antibodies, and, currently,most commercial therapeutic antibodies are IgG1 format. To constructhumanized 2A2 IgG1 in a mammalian expression vector, pOptiVEC and pcDNA3.3 (Invitrogen) vectors were used.

Vector for the Expression of Humanized 2A2 IgG1 in Mammalian Cells

The exemplary vector contains the human cytomegalovirus (CMV)immediate-early promoter/enhancer for high-level expression ofrecombinant proteins in a wide range of mammalian cells. To constructionof humanized 2A2 IgG1, human variable light and heavy chain each withthree CDRs of mouse 2A2 were synthesized and these two DNA fragmentswere linked to the human constant light and heavy chain by PCR. Finally,the humanized 2A2 light chain was cloned into pcDNA3.3 TOPO, andhumanized 2A2 heavy chain was cloned into the pOptiVEC TOPO antibodyexpression vector. Sequences of human 2A2 IgG1 are shown in FIGS. 2A-B,which indicated that first amino acid (Arginine, red color shading) ofhuman constant light chain was missed during construction of wholehumanized light chain. After construction of these human 2A2 Abexpression vectors, the DNA plasmids were co-transfected intoCHO-derived, DHFR-negative DG44 cells to create a stable cell line thatproduces 2A2 hIgG1 antibody.

Development of Stable Cell Lines for Antibody Production

To obtain cell lines that produce high levels of antibody, a pool ofstably-transfected cells were selected by performing two rounds ofselection using CD OptiCHO medium and CD OptiCHO medium with 500 μg/mlof Geneticine, followed by MTX genomic amplification selection and tworounds of single cell clonal selection in semi-solid media in a 96-wellplate. Antibody expression levels were screened by ELISA assayquantification and selected h2A2IgG1-CHO cell (G3A10, C5G6 and D5F11)lines were slowly scaled up.

Example 2: Generation of Additional Anti-Ceramide Antibodies

A panel of monoclonal antibodies was generated for use inrepeat-administration studies in mice to study immunogenicity. Screeningof hybridomas to select anti-ceramide Mabs was performed by ELISA, usingthe antigen (Omega-COOH C16-ceramide coupled to albumin). Positive hitswere counterscreened against both BSA and Omega-COOH C16-dihydroceramidecoupled to albumin. Biologic testing of Mabs (in vitro inhibition ofJurkat cell apoptosis, in vivo inhibition of Radiation GI Syndrome) wasthen performed. Clones designated 9H10, 9H11, 9A2, 7B10, 6B5 and 6C8were selected for testing and all but 9A2 demonstrated biologic activityin vitro. As shown in Table 1, a panel of clones preferentially bindC16:0 carboxyceramide-BSA. Ag1 is C16:0 carboxyceramide-BSA coated @ 300ng/well, Ag2 is C-16:0 dihydro-carboxyceramide-BSA coated @ 300 ng/welland Ag3 is Free BSA (Sigma A6003) coated @ 300 ng/well.

TABLE 1 Anti-Ceramide mAbs Ag1 OD AG1 OD Ag2 OD Ag3 OD Clone #(IgG(gamma)) (IgM(Mμ) (gamma) (gamma) 6B5 2.201 0.054 0.049 1.295 7E81.530 0.045 0.077 0.906 8H8 3.000 0.103 0.098 3.000 9A2 3.000 0.0550.078 3.000 7B10 0.080 0.180 0.078 0.077 9H10 0.052 0.230 0.064 0.0959H11 0.045 0.343 0.047 0.105 6C8 0.192 0.039 0.049 0.098 NC 0.047 0.0660.048 0.096 PC 3.000 0.067 0.070 3.000 NC = 50% of culture media + 50%of 5% milk/PBS PC = Cardiac Serum Mouse #1 @ 1:1K

Clones 6B5 and 6C8 were identified as IgG while clones 7B10, 9H10 and9H11 were identified as IgM. Additional data for those identified as IgGis found in Table 2. Ag1 is C16:0 carboxyceramide-BSA coated @ 500ng/well in sodium bicarbonate, Ag2 is C-16:0 dihydro-carboxyceramide-BSAcoated @ 500 ng/well in sodium bicarbonate and Ag3 is Free BSA (SigmaA6003) coated @ 500 ng/well in sodium bicarbonate.

TABLE 2 Concentration Curve of Anti-Ceramide mAbs Clone #/ Ag1 AG1 Ag2Ag2 Ag3 Ag3 Ab dilution (gamma) (Mμ) (gamma) (Mμ) (gamma) (Mμ) 6B5 0.6390.072 0.150 (0.99 mg/ml) 10 μg/ml 6B5 0.399 0.089 0.119 (0.99 mg/ml) 1μg/ml 6B5 0.147 0.072 0.079 (0.99 mg/ml) 0.1 μg/ml 6C8 0.119 0.073 0.062(0.79 mg/ml) 10 μg/ml 6C8 0.057 0.074 0.057 (0.79 mg/ml) 1 μg/ml 6C80.052 0.068 0.056 (0.79 mg/ml) 0.1 μg/ml NC 0.053 0.062 0.048 0.0520.060 0.056 PC 1.426 0.218 0.094 0.070 0.350 0.127 NC = 5% milk-PBS PC =CERM01 Tail Bleed Serum Mouse #1 @ 1:1K

FIG. 3 shows that clones 9H10, 9H11, 7B10, 6B5 and 6C8 demonstratebiologic activity in vitro.

Positive clones were screened for biologic activity by exposing Jurkatcells to 10 Gy penetrating radiation. Monoclonal antibodies (Mabs) addedto culture medium at indicated doses just prior to IR and the cells werefixed after 16 hr incubation. Apoptosis was quantified by HOESCHSTbisbenzimide stain and morphologic examination. The results are shown inFIG. 4.

Crypt lethality was studied on clones 7B10 (IgM), 6B5 (IgG), and 6C8(IgG). FIG. 5 shows that all dose-dependently inhibited crypt lethalitywhen administered 15 minutes prior to the 15 Gy IR.

The CDRs of 6B5, 6C8, 7B10, 9H10 and 9H11 were sequenced. Sequence datarevealed significant homology amongst these Mabs, as well as with theCDRs of 2A2 (generated via an alternative immunization/screeningprotocol). IgMs appear to have even greater homology amongst each otherand 2A2. In the same way the two IgG are most similar to each other.FIG. 6 shows a sequence alignment of the six murine antibody heavy andlight chain variable region sequences, as well as the sequence forhumanized h2A2 (derived from m2A2), as well as a depiction of a computergenerated consensus sequence. In CDR1 of the heavy chain variableregion, each of the antibodies comprise 10 amino acids comprising a Glyin the 1st position from the N-terminal, a Tyr or Phe in the 2ndposition from the N-terminal, a Phe or Leu in the 4th position from theN-terminal, and a Thr or Ser in the 5th position from the N-terminal anda His or Asn in the 10th position from the N-terminal. In CDR2 of theheavy chain variable region, each of the antibodies comprise 16-17 aminoacids comprising a Asn or Ile in the 2nd position from the N-terminal, aPhe or Ser in the 4th position from the N-terminal, a Thr in the 9thposition from the C-terminal, a Tyr in the 7th position from theC-terminal, an Asn or Arg in the 6th position from the C-terminal, a Lysor Ala in the 4th position from the C-terminal and a Phe in the 3rdposition from the C-terminal. In CDR3 of the heavy chain variable regionof the murine antibodies, each of the antibodies comprise 7 to 11 aminoacids comprising a Tyr or Thr at the 4th position from the N-terminal.In CDR1 of the light chain variable region, each of the antibodiescomprise 10-16 amino acids comprising an Ala or Ser in the 2nd positionfrom the N-terminal, a Ser in the 3rd position from the N-terminal, aSer or Asp in the 5th position from the N-terminal, and a Tyr, Ser orPhe in the 3rd position from the C-terminal. In CDR2 of the light chainvariable region, each of the antibodies comprise 7 amino acidscomprising a Ser or Asn in the 3rd position from the N-terminal, a Lysor Ser in the 5th position from the N-terminal and a Ser or Asp in the7th position from the N-terminal. In CDR3 of the light chain variableregion, each of the murine antibodies comprise 9 amino acids comprisinga Gln, Leu or Trp in the 1st position from the N-terminal, a Gln in the2nd position from the N-terminal, a Pro in the 7th position from theN-terminal and a Thr in the 9th position from the N-terminal.

Also shown in FIG. 6 are anti-ceramide consensus sequence heavy chainand light chain CDRs based upon the sequence information derived fromthe CDR sequences of 6B5, 6C8, 7B10, 9H10 and 9H11. The presentinventors have surprisingly found that the CDR regions of the light andheavy chain variable regions of the anti-ceramide antibodies havecertain conserved amino acid residues. In some embodiments, a consensussequence determined from the sequence of two or more of theanti-ceramide antibodies 6B5, 6C8, 7B10, 9H10, 9H11 and 2A2 can be usedto generate an scFv antibody comprising consensus CDRs, consensusvariable regions, or variable regions comprising at least about 80%, 90%or 95% sequence identity with a consensus variable region sequence.

Example 3: Generation of Anti-Ceramide scFv

Based on Mab efficacy data, the CDRs of 6B5 were chosen (along with 2a2)to be engineered into a single-chain Fv. Two single chain (sc) Fvconstructs were engineered to express the scFv and provide purified scFvfor efficacy testing. 6B5 scFv was readily expressed and purified.Biologic testing of scFv, as with Mabs, was performed using in vitroinhibition of Jurkat cell apoptosis and in vivo inhibition of RadiationGI Syndrome. FIG. 5 shows that 6B5 IgG protects against crypt death viathe microcolony assay. FIG. 7 shows scFv inhibits jurkat cell apoptosis.FIG. 8 shows that 6B5 scFv protects against GI crypt depletion in vivowhen administered 15 minutes prior to 15 Gy exposure. FIG. 9 shows that6B5 scFv mitigates against GI crypt depletion in vivo when administered24 hours after 15 Gy exposure.

Anti-Ceramide scFv Protects Mice from Lethal Acute Graft-Versus-HostDisease

C57BL/6 mice (MHC H2^(b) haplotype) were administered saline, 50 mg/kghumanized anti-ceramide h2A2 or 7.5 mg/kg anti-ceramide scFv 6B5 via theindicated route of administration and dosing schedule. Dosing began 15min prior to 1100 cGy split-dose total-body irradiation (TBI). Micesubsequently received an allogeneic bone marrow transplantation 16-20hours post TBI consisting of 5×10⁶ allogeneic bone marrow (BM) or BM and2×10⁶ allogeneic CD5+ naïve T cells from B10.BR donor mice (MHC H2^(k2)haplotype). Mice were monitored daily for survival. Data represents Day10 survival, determined to be representative of 90 day survival. Anintravenous route of administration with saline on a dosing schedule of0, 4, and 8 days resulted in 30% survival after 10 days. An intravenousroute of administration with h2A2 monoclonal antibody on a dosingschedule of 0, 4, and 8 days resulted in 100% survival after 10 days(p<0.001 vs. saline control). An intravenous route of administrationwith scFv on a dosing schedule of 0, 4, and 8 days resulted in 60%survival after 10 days (p<0.05 vs. saline control). A subcutaneous routeof administration with saline on a dosing schedule of 0, 2, 4, 6 and 8days resulted in 0% survival after 10 days. A subcutaneous route ofadministration with saline on a dosing schedule of 0, 2, 4, 6 and 8 daysresulted in 100% survival after 10 days (p<0.001 vs. saline control).

As shown in FIG. 10, anti-ceramide scFV protects intestinal crypts in adose-dependent manner. As shown in FIG. 11, anti-ceramide scFv retainsefficacy when administered via alternative injections. As shown in FIG.12, anti-ceramide h2A2 and scFv protect and mitigate the lethal effectsof Radiation GI Syndrome. As shown in FIG. 13, anti-ceramide scFvprotects mice from lethal acute graft-versus-host disease. As shown inFIG. 14, anti-ceramide scFv protects mouse intestinal stem cells duringlethal acute graft-versus-host disease. As shown in FIG. 15,anti-ceramide h2A2 and scFv increase retention of CD4+ and CD8+lymphocytes within the mesentery lymph nodes (FIG. 15).

The above description is for the purpose of teaching the person ofordinary skill in the art how to practice the present invention, and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentinvention, which is defined by the following claims. The claims areintended to cover the components and steps in any sequence which iseffective to meet the objectives there intended, unless the contextspecifically indicates the contrary.

1.-26. (canceled)
 27. An anti-ceramide antibody or antigen-bindingfragment thereof comprising a variable heavy chain (V_(H)) and avariable light chain (V_(L)), wherein the V_(H) comprises a heavy chainvariable region CDR1 comprising the sequence GYTFTDHTIH (SEQ ID NO: 1),a heavy chain variable region CDR2 comprising the sequenceYNYPRDGSTKYNEKFKG (SEQ ID NO: 2), and a heavy chain variable region CDR3comprising the sequence GFITTVVPSAY (SEQ ID NO: 3), and the V_(L)comprises a light chain variable region CDR1 comprising the sequenceRASKSISKYLA (SEQ ID NO: 4), a light chain variable region CDR2comprising the sequence SGSTLQS (SEQ ID NO: 5), and a light chainvariable region CDR3 comprising the sequence QQHNEYPWT (SEQ ID NO: 6).28. An anti-ceramide antibody or antigen-binding fragment thereofcomprising a heavy chain variable region sequence that is at least about90% identical to a heavy chain variable region sequence comprising SEQID NO: 7; and/or a light chain variable region sequence that is at leastabout 90% identical to a light chain variable region sequence comprisingSEQ ID NO:
 8. 29. An anti-ceramide antibody or antigen-binding fragmentthereof comprising a heavy chain variable region sequence ofQVQLQQSDAELVKPGASVKISCKVSGYTFTDHTIHWMKQRPEQGLEWIGYNYPRDGSTKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCAKGFITTVVPSAYWGQGTLV TVSA (SEQ IDNO: 48) and/or a light chain variable region sequence ofDVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPWTFGGGTKLEIK (SEQ ID NO: 8).
 30. Theanti-ceramide antibody or antigen-binding fragment of claim 27, whereinthe antibody is selected from the group consisting of a monoclonalantibody, a chimeric antibody, a humanized antibody, a human antibodyand a scFv.
 31. The anti-ceramide antibody of claim 30, wherein theantibody is a scFv.
 32. An anti-ceramide antibody or antigen-bindingfragment thereof that binds to the same antigenic determinant as theanti-ceramide antibody or antigen-binding fragment of claim
 27. 33. Amethod of inhibiting apoptosis in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of theanti-ceramide antibody or antigen-binding fragment of claim
 27. 34. Themethod of claim 33, wherein the anti-ceramide antibody is a scFvantibody.
 35. The method of claim 33, wherein the apoptosis isassociated with a disease selected from the group consisting of graftversus host disease, radiation disease, GI syndrome, and autoimmunedisease.
 36. The method of claim 35, wherein the disease is radiationdisease or GI syndrome and the anti-ceramide antibody or antigen-bindingfragment is administered before the subject is exposed to radiation. 37.The method of claim 35, wherein the disease is graft versus host diseaseand the anti-ceramide antibody or antigen-binding fragment isadministered before the subject receives a transplant.
 38. The method ofclaim 37, wherein the transplant is a bone marrow transplant.
 39. Themethod of claim 33, wherein the anti-ceramide antibody orantigen-binding fragment is administered intravenously, intramuscularly,intraperitoneally, intracerobrospinally, subcutaneously,intrasynovially, intrathecally, orally, topically, or via inhalation.40. A method for mitigating apoptosis in a subject with GI syndromecomprising: administering to the subject an effective amount of theanti-ceramide antibody or antigen binding fragment of claim 27, afterthe subject is exposed to penetrating radiation.
 41. The method of claim40, wherein the anti-ceramide antibody is an scFv antibody.
 42. Themethod of claim 40, wherein the anti-ceramide antibody or antigenbinding fragment is administered immediately after the subject isexposed to penetrating radiation.
 43. The method of claim 40, whereinthe anti-ceramide antibody or antigen binding fragment is administeredwithin 24 hours after the subject is exposed to penetrating radiation.44. A method for inhibiting apoptosis in a subject with GvHD comprising:administering to the subject an effective amount of the anti-ceramideantibody or antigen binding fragment of claim 27, either before thesubject receives a transplant or after the subject receives a transplantprior to the onset of GvHD.
 45. The method of claim 44, wherein thetransplant is a bone marrow transplant.
 46. The method of claim 44,wherein the anti-ceramide antibody is a scFv antibody.